The Arabidopsis thaliana mutants coi1-1(Xie et al.,1998),coi1-2(Xu et al., 2002),JAZ1D3A(Thines et al.,2007),myc2-2(Boter et al.,2004),jin1-2 (Lorenzo et al.,2004),myc3(GK445B11)(Fernández-Calvo et al.,2011), myc4(GK491E10)(Fernández-Calvo et al.,2011),jin1-2myc3myc4 (Fernández-Calvo et al.,2011),eto1-1(Guzmán and Ecker,1990),ctr1-1 (Kieber et al.,1993),ein2-1(Alonso et al.,1999),hls1-1(Lehman et al., 1996),ein3-1(Chao et al.,1997),eil1-3/Salk_049679(Binder et al.,2007), myb21myb24myb57(Cheng et al.,2009),and gl3egl3tt8(Qi et al.,2011) were previously described.The higher order mutants myc2-2myc3, myc2-2myc4,myc2-2myc3myc4,ein3-1eil1-3,coi1-2ein3-1eil1-3, coi1-2hls1-1,myc2-2ein3eil1-3,and jin1-2myc3myc4ein3-1eil1-3were generated by genetic crosses using standard techniques.
The Arabidopsis seeds were sterilized with20%bleach,plated on Murashige and Skoog(MS)medium,chilled at4°C for3d,and then transferred to a growth room under a16-h(20to24°C)/8-h(16to19°C) light/dark photoperiod.For hook phenotype analysis,seeds were ster-ilized,chilled,and transferred to a growth chamber at22°C in the dark for 4d.Nicotiana benthamiana was grown in a growth room under a16-h (25to28°C)/8-h(22to25°C)light/dark cycle.
BiFC Assay
For the BiFC assays,the full-length coding sequence(CDS)of Arabi-dopsis EIN3,EIL1,MYC2,MYC3,and MYC4were cloned into the binary nYFP or cYFP vector through the Gateway system(Invitrogen)(Qi et al., 2011).Primer pairs used for the generation of constructs are listed in Supplemental Table1.BiFC assays were performed as previously de-scribed(Qi et al.,2011).Equal concentrations and volumes of re-suspended Agrobacterium tumefaciens strain GV3101harboring the indicated nYFP or cYFP vectors in in?ltration buffer(0.2mM acetosyr-ingone,10mM MgCl
2
,and10mM MES)were mixed and coin?ltrated into leaves of N.benthamiana using a needleless syringe.Two days after in?ltration,the YFP signal was observed using a Zeiss confocal micro-scope(LSM710).Four hours before observation,100m M MG132was in?ltrated into the leaves of N.benthamiana.
Pull-Down Assay
MBP-MYC2(Chen et al.,2011)and MBP proteins were puri?ed from Es-cherichia coli using MBP af?nity chromatography according to Qi et al. (2011).The full-length CDS of EIN3was cloned into the modi?ed pCam-bia1300vector under the control of35S promoter for fusion with three?ag tags to generate?ag-EIN3.Agrobacterium strain harboring?ag-EIN3was in?ltrated into N.benthamiana leaves.After50h,5g of N.benthamiana leaves transiently expressing?ag-EIN3were harvested for total protein extraction in RB buffer(100mM NaCl,50mM Tris-Cl,pH7.8,25mM imidazole,0.1%[v/v]Tween20,10%[v/v]glycerol,EDTA-free complete miniprotease inhibitor cocktail,and20mM2-mercaptoethanol).The ex-tracted total protein was concentrated in centrifugal?lter tubes(Millipore)to 400μL.Coomassie Brilliant Blue was used to con?rm the protein amount. About50μg of puri?ed MBP and MBP-MYC2was incubated with120m L of amylose resin beads for2h at4°C.These amylose resin beads were then washed?ve times with1mL of RB buffer and incubated with200m L of concentrated total proteins containing?ag-EIN3for2h at4°C.After washing?ve times with1mL RB buffer,the mixture was resuspended in SDS loading buffer,boiled for5min,separated on15%SDS-PAGE,and immunoblotted using1:1000dilution for anti-?ag antibody(Abmart).
Co-IP
N.benthamiana leaves were in?ltrated with Agrobacterium strains har-boring?ag-EIN3,?ag-EIN3with myc-MYC2(Zhai et al.,2013),or?ag-EIN3with myc-COI1(Yan et al.,2013).Two days after in?ltration,3g of agroin?ltrated leaves for each combination was collected and homoge-nized in Co-IP buffer containing50mM Tris-HCl,pH7.5,100mM NaCl, 2mM DTT,0.1%Tween20,1mM phenylmethylsulfonyl?uoride,50mM MG132,and complete protease inhibitor cocktail(Roche)and centrifuged twice at16,000g at4°C.The supernatant was concentrated to400m L and incubated with the agarose-conjugated anti-myc matrix(Abmart)for2h (4°C,with rotation),then washed three times with1mL of immunopre-cipitation buffer.After denaturation in100m L of SDS loading buffer,the samples were loaded into15%SDS-PAGE gels,subjected to gel elec-trophoresis,transferred to polyvinylidene?uoride membranes(Millipore), and immunoblotted with anti-?ag antibody(Abmart)and anti-myc anti-body,respectively.
Protoplast Transfection Assay
For the transient transcriptional activity assay,constructs harboring the LUC gene under the control of the;1500-bp or;1523-bp promoter sequences of HLS1or ERF1,respectively,in the pGreenII0800-LUC vector were generated as reporters(Hellens et al.,2005).The renilla luciferase(REN)gene under the control of35S promoter in the pGreenII 0800-LUC vector was used as the internal control.The CDS sequences of EIN3,EIL1,and MYC2were cloned into the pGreenII62-SK vector under the control of the35S promoter and were used as effectors.All primers used for making these constructs are listed in Supplemental Table1. Arabidopsis mesophyll protoplast was prepared and transfected as previously described(Yoo et al.,2007).The?re?y LUC and REN activities
MYC2-EIN3Mediates JA-ET Antagonism275
were measured using the Dual-Luciferase Reporter Assay System (Promega).LUC/REN ratios were presented.
For transient expression assay,the CDS of MYC2fused with GAL4DB (GAL4DB-MYC2)under the control of35S promoter was used as effector. The GUS gene under the control of four copies of upstream GAL4DNA binding sites[GAL4(4x)-D1-3(4x)]was used as the reporter(Tiwari et al.,2001; Zhu et al.,2008).The?re?y LUC gene under the control of35S promoter was the internal control.Primers used for plasmid construction are shown in Supplemental Table1.The preparation and subsequent transfection of Arabidopsis mesophyll protoplasts were performed as described previously(Yoo et al.,2007).The GUS/LUC ratios were presented. Infection with Pathogen
Detached leave from3-week-old plant were inoculated with5m L spores of Botrytis cinerea(SCL2-4,isolated from tomato in2011,Shanghai)(Song et al.,2013b)(105spores/mL)suspended in potato dextrose broth(with potato dextrose broth alone as the control),placed in Petri dishes with0.8% agar,and covered with lids.The lesion diameter from eight leaves for each genotype exhibiting disease symptoms was measured2d after inoculation. Insect Defense Assay with Spodoptera exigua
Newly hatched S.exigua larvae were placed on3-week-old plants(10-h-light/14-h-dark photoperiod)of each genotype for7d of feeding.Ten surviving larvae were weighted as one sample to obtain one datum for average weight.Fifty surviving larvae(?ve independent samples in total) for each genotype in each biological experiment were used.The ex-periment was repeated for three biological replicates.
Quantitative Real-Time PCR
For Figures1B,3B,and6B,Arabidopsis seedlings were grown on MS medium at22°C in the dark for4d.For Figures1C,3C,and6C,seedlings were grown on MS medium at22°C in the dark for4d and then were treated with mock,100m M methyl jasmonate(MeJA),100m M ACC,or 100m M MeJA plus100m M ACC for6h.For Figures7C,9A,and9B, seedlings were grown on MS medium for12d under a16-h(20to24°C)/ 8-h(16to19°C)light/dark photoperiod and then were treated with mock or100m M MeJA for6h.These materials were harvested for RNA ex-traction and subsequent reverse transcription.Real-time PCR analyses were performed using the ABI7500real-time PCR system with the RealMasterMix(SYBR Green I)(Takara)as described previously(Qi et al., 2011).The primers for real-time PCR analysis are presented in Supplemental Table2online.ACTIN8was used as the internal control.The experiment was repeated for three biological replicates.
Accession Numbers
The Arabidopsis Genome Initiative numbers for genes mentioned in this article are as follows:COI1(AT2G39940),MYC2(AT1G32640),MYC3(AT5G46760), MYC4(AT4G17880),JAZ1(AT1G19180),ETO1(At3g51770),CTR1 (AT5G03730),EIN2(AT5G03280),EIN3(AT3G20770),EIL1(AT2G27050), ERF1(AT3G23240),HLS1(AT4G37580),PDF1.2(AT5G44420),MYB21 (At3g27810),MYB24(At5g40350),MYB57(At3g01530),TT8(AT4G09820), GL3(AT5G41315),EGL3(AT1G63650),VSP1(AT5G24780),VSP2 (AT5G24770),TAT3(AT2G24850),ORA59(AT1G06160),CYP79B3 (At2g22330),BCAT4(At3g19710),BAT5(At4g12030),and ACTIN8 (AT1G49240).
